Tuberculosis is resurgent in most of the world fueled by drug resistance, AIDS, poverty and mobility. The goal of this project is to produce an improved vaccine. It is based on our observations that an antigen 85A deletion mutant of MTB strain H37Rv (Ag85A-) is markedly attenuated and has increased antigen presenting capability. Our rationale has three components: First, the most effective immunity known against tuberculosis is provided by prior infection with MTB itself. Consequently, we propose that an appropriately attenuated MTB will approach the natural limit of efficacy of vaccines for tuberculosis. Second, MTB inhibits phagosome-lysosome (P-L) fusion. Preliminary data demonstrates that deletion of the Ag85A gene of MTB restores P-L fusion, enhances antigen presentation and probably increases the immunogenicity of MTB. Third, since MTB is a clonal organism with no demonstrated ability for horizontal transfer of genes, the safety of attenuated MTB can be assured. The specific aims are: 1) Evaluate the efficacy and safety of the Ag85A-mutant as a vaccine against tuberculosis. The efficacy (ability to limit primary infection and dissemination) will be investigated by single or multiple immunizations of C57BL/6 mice, outbred mice and guinea pigs prior to aerosol challenge with virulent MTB. Safety will be evaluated by infection of a spectrum of animals including guinea pigs that are naturally more susceptible to disease, genetically heterogeneous outbred mice, immunocomormized SCID mice and steroid treated mice. Safety will be evaluated in terms the capacity of the vaccine organism to produce disease and its effect on persistence and pathogenicity of wild type MTB following challenge. 2) Introduce additional deletion mutations into the Ag85A- strain to improve safety while maintaining immunogenicity. We anticipate that any vaccine with prolonged survival in tissue may produce disease in immunosuppressed people. Consequently, attempts will be made to produce a double knockout mutant MTB that retains the immunogenicity of Ag85A- and is unable to survive in tissue. The 16 kDa alpha crystallin protein gene and nitrate reductase gene will be targeted. We anticipate that a safe live attenuated MTB vaccine with enhanced immunogenicity will prove valuable in combating adult pulmonary tuberculosis as well primary disease.